1. Field of the Invention
The present invention relates generally to the field of fat metabolism and weight control. More specifically, the present invention relates to the role of the ACC2 isoform of acetyl-CoA carboxylase in regulating fatty acid accumulation and oxidation and to transgenic mice deficient for the ACC2 isoform.
2. Description of the Related Art
Acetyl-CoA carboxylase (ACC), a biotin-containing enzyme, catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA, an intermediate metabolite that plays a pivotal role in the regulation of fatty acid metabolism (1-3). It has been found that malonyl-CoA is a negative regulator of carnitine palmitoyltransferase I (CPTI, a component of the fatty-acid shuttle system (4,5) that is involved in the mitochondrial oxidation of long-chain fatty acids. This finding provides an important link between two opposed pathways-fatty-acid synthesis and fatty-acid oxidation. Thus, it is possible to interrelate fatty acid metabolism with carbohydrate metabolism through the shared intermediate acetyl-CoA, the product of pyruvate dehydrogenase. Consequently, the roles of malonyl-CoA in energy metabolism in lipogenic (liver and adipose) and non-lipogenic (heart and muscle) tissues has become the focus of many studies (4-11).
In prokaryotes, acetyl-CoA carboxylase is composed of three distinct proteinsxe2x80x94the biotin carboxyl carrier protein, the biotin carboxylase, and the transcarboxylase (12). In eukaryotes, however, these activities are contained within a single multifunctional protein that is encoded by a single gene. In animals, including humans, there are two isoforms of acetyl-CoA carboxylase expressed in most cells, ACC1 (Mrxcx9c265,000) and ACC2 (Mrxcx9c280,000), which are encoded by two separate genes and display distinct tissue distribution (2-6, 13-17). Both ACC1 and ACC2 produce malonyl-CoA, which is the donor of the xe2x80x9cC2-unitsxe2x80x9d for fatty acid synthesis and the regulator of the carnitine palmitoyl-CoA shuttle system that is involved in the mitochondrial oxidation of long-chain fatty acids (4, 5, 18). Hence, acetyl-CoA carboxylase links fatty acid synthesis and fatty acid oxidation and relates them with glucose utilization and energy production because acetyl-CoA, the substrate of the carboxylases, is the product of pyruvate dehydrogenase. This observation, together with the finding that ACC1 is highly expressed in lipogenic tissues such as liver and adipose and that ACC2 is predominantly expressed in heart and skeletal muscle (3,14,17,19), opened up a new vista in comparative studies of energy metabolism in lipogenic and fatty acid-oxidizing tissues.
Diet, especially a fat-free one, induces the synthesis of ACC""s and increases their activities. Starvation or diabetes mellitus represses the expression of the ACC genes and decreases the activities of the enzymes. Earlier studies addressed the overall activities of the carboxylases with specific differentiation between ACC1 and Acc2. Studies on animal carboxylases showed that these enzymes are under long-term control at the transcriptional and translational levels and short-term regulation by phosphorylation/dephosphorylation of targeted Ser residues and by allosteric modifications induced by citrate of palmitoyl CoA (16,20-25). Several kinases have been found to phosphorylate both carboxylases and to reduce their activities. In response to dietary glucose, insulin activates the carboxylases through their phosphorylation. Starvation and/or stress lead to increased glycogen and epinephrin levels that inactivate the carboxylases through phosphorylation (20-25). Experiments with rats undergoing exercises showed that their malonyl CoA and ACC activities in skeletal muscle decrease as a function of exercise intensity thereby favoring fatty acid oxidation. These changes are associated with an increase in AMP-kinase activity (25-28). The AMP-activated protein kinase (AMPK) is activated by a high level of AMP concurrent with a low level of ATP through mechanism involving allosteric regulation and phosphorylation by protein kinase (AMP kinase) in a cascade that is activated by exercise and cellular stressors that deplete ATP (7-10). Through these mechanisms, when metabolic fuel is low and ATP is needed, both ACC activities are turned off by phosphorylation, resulting in low malonyl-CoA levels that lead to increase synthesis of ATP through increased fatty acid oxidation and decreased consumption of ATP for fatty acid synthesis.
Recently, it was reported that the cDNA-derived amino acid sequences of human ACC1 and ACC2 share 80% identity and that the most significant difference between them is in the N-terminal sequence of ACC2 (3,13). The first 218 amino acids in the N-terminus of ACC2 represents a unique peptide that includes, in part, 114 of the extra 137 amino acid residues found in this isoform (14). Polyclonal antibodies raised against the unique ACC2 N-terminal peptide reacted specifically with ACC2 proteins derived from human, rat, and mouse tissues. These findings made it possible to establish the subcellular localization of ACC1 and ACC2 (14) and to later demonstrate that ACC2 is associated with the mitochondria and that the hydrophobic N-terminus of the ACC2 protein plays an important role in directing ACC2 to the mitochondria (6). ACC1, on the other hand, is localized to the cytosol.
Although these findings and the distinct tissue distribution of ACC1 and ACC2 suggest that ACC2 is involved in the regulation of fatty acid oxidation and that ACC1 is involved in fatty acid synthesis primarily in lipogenic tissues, they do not provide direct evidence that the products of the genes ACC1 and ACC2 have distinct roles.
The prior art is deficient in an understanding of the separate roles of ACC1 and ACC2 have in the fatty acid metabolic pathway. The prior art is also deficient in transgenic knockout mice generated to lack ACC2 and methods of using these transgenic mice. The present invention fulfills this long-standing need and desire in the art.
Malonyl-CoA (Ma-CoA), generated by acetyl-CoA carboxylases ACC1 and ACC2, is key metabolite in the regulation of fatty acid (FA) metabolism. ACC1xe2x88x92/xe2x88x92 mutant mice were embryonically lethal, possibly due to lack of xe2x80x9cC2-unitsxe2x80x9d for the synthesis of fatty acid needed for biomembrane synthesis. ACC2xe2x88x92/xe2x88x92 mutant mice bred normally and had normal life spans. ACC2xe2x88x92/xe2x88x92 mice fed normal diet did not accumulate fat in their livers as did the wild-type mice and overnight fasting resulted in 5-fold increase in ketone bodies production, indicating higher fatty acid oxidation. ACC1 and fatty acid synthase activities and malonyl-CoA contents of the livers of the ACC2xe2x88x92/xe2x88x92 and ACC2+/+ mice were the same, indicating that fatty acid synthesis is unperturbed yet the malonyl-CoA was not available for the inhibition of the mitochondrial fatty acid shuttle system, hence fatty acid oxidation was relatively high.
Absence of ACC2 resulted in 30- and 10-fold lower malonyl-CoA contents of muscles and heart, respectively. Fatty acid oxidation in the ACC2xe2x88x92/xe2x88x92 soleus muscles was 30% higher than that of ACC2+/+ mice. Addition of insulin did not affect fatty acid oxidation in the ACC2xe2x88x92/xe2x88x92 soleus muscle, but, as expected, it did reduce fatty acid oxidation by 50% in the wild-type soleus muscle compared to that of the mutant. Isoproterenol, an analog of glucagon, had little effect on fatty acid oxidation in the muscle of the ACC2xe2x88x92/xe2x88x92 mice but caused 50% increase in fatty acid oxidation in the soleus muscle. The higher fatty acid oxidation in the mutant mice resulted in 50% reduction of fat storage in the adipose tissue compared to that of the wild-type mice. These results are valuable to an understanding and control of fatty acid metabolism and energy homeostasis in normal, diabetic, and obese animals.
In one embodiment of the present invention, there is provided a transgenic mouse having a mutation in an endogenous gene for the ACC2 isoform of acetyl-CoA carboxylase that inactivates the protein. The ACC2 gene may be mutated by deleting one or more exons of the gene, which may be replaced by heterologous DNA sequences such as an HPRT expression cassette. In a preferred embodiment, an exon encoding a biotin-binding motif of ACC2 is replaced with an HPRT expression cassette. These mice exhibit a phenotype consisting of a reduction in malonyl-CoA levels in skeletal muscle and heart, unrestricted fat oxidation, and reduced fat accumulation in the liver and fat storage cells. The transgenic mice consume more food than wild type mice but remain lean.
In yet another embodiment of the present invention, there is provided a method of screening for an inhibitor of ACC2 isoform activity consisting of the step of administering potential inhibitors to wild type mice and screening for mice which exhibit the same phenotype of the ACC2xe2x88x92/xe2x88x92 transgenic mice.
In yet another embodiment of the present invention, there is provided an ACC2 inhibitor identified by the above method. This inhibitor may be incorporated into a pharmaceutical composition to be administered to individuals for purposes of augmenting fatty acid oxidation and inhibiting fat accumulation to promote weight loss or maintenance.
In a further embodiment of the instant invention, a method is described for obtaining a purified preparation of ACC1 protein totally free of the ACC2 isoform by purifying ACC1 from the Acc2xe2x88x92/xe2x88x92 transgenic mice.
In another embodiment of the instant invention, a method is provided for obtaining improved antibodies against ACC2 by generating the antibodies in the Acc2xe2x88x92/xe2x88x92 transgenic mice.
In yet another embodiment of the instant invention, cell lines derived from the Acc2xe2x88x92/xe2x88x92 transgenic mice are provided. Cell lines derived from muscle, heart, adipose cells, and liver cells are expected to be especially useful in bioassays and drug targeting studies.
In yet another embodiment of the present invention, a method of screening for agonists and antagonists of ACC2 is provided. This method comprises the steps of administering candidate compounds Acc2xe2x88x92/xe2x88x92 cell lines and to cell lines derived from wild type mice followed by experiments to detect alterations in cellular activity. A compound that specifically acts on ACC2 will alter cellular activity in wild type cells but have no effects on Acc2xe2x88x92/xe2x88x92 cells. Cellular activities that may be monitored include mRNA expression, protein expression, protein secretion, and lipid metabolism.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the embodiments of the invention given for the purpose of disclosure.